Antiviral activities of extracts and selected pure constituents of Ocimum basilicum

Development of broad-spectrum enterovirus antivirals based on quinoline scaffold

Backgroundhyaluronic acid (HA), a non-sulphated glycosaminoglycan, is present in synovial fluid, vitreous humour serum and many connective tissues. Pharmaceutical preparations of HA are used in clinical practice for wound healing, joint pain, kerato-conjunctivitis, asthma, mouth care, oesophageal-reflux, and gastritis. Moreover, it is used as a filler to counteract ageing and facial lipoatrophy. Our study aims at investigating the in vitro antiviral activity of a high molecular weight HA.Methodsthe MTT test was used to rule out the potential toxic effects of HA on the different cell lines used in the antiviral assays. The antiviral activity of HA against Coxsackievirus B5, Herpes Simplex Virus-1, Mumps Virus, Adenovirus-5, Influenza Virus A/H1N1, Human Herpesvirus-6, Porcine Parvovirus, Porcine Reproductive and Respiratory Syndrome Virus was assessed by virus yield assays.Resultsthe most effective inhibition was observed against Coxsackievirus B5, with 3Log reduction of the virus yield at 4 mg/ml, and a reduction of 3.5Log and 2Log, at 2 mg/ml and 1 mg/ml, respectively: the selectivity index was 16. Mumps virus was highly inhibited too showing a reduction of 1.7Log at 1 mg/ml and 1Log at 4 mg/ml and 2 mg/ml (selectivity index = 12). The selectivity index for Influenza Virus was 12 with the highest inhibition (1Log) observed at 4 mg/ml. Herpes Simplex Virus-1 and Porcine Parvovirus were mildly inhibited, whereas no antiviral activity was observed with respect to Adenovirus-5, Human Herpesvirus-6, Porcine Reproductive and Respiratory Syndrome Virus. No HA virucidal activity was ever observed against any of the viruses tested. Kinetic experiments showed that both Coxsackievirus B5 and Herpes simplex virus-1 replication were consistently inhibited, not influenced by the time of HA addition, during the virus replication cycle.Conclusionsthe spectrum of the antiviral activity exhibited by HA against both RNA and DNA viruses, known to have different structures (with or without envelope) and replication strategies, suggests a non specific mechanism of action, probably involving cell membrane-virus interaction steps. The results of the kinetic experiments support this hypothesis.

Objective To investigate the serotypes of human enterovirus B (HEV-B) species causing hand, foot and mouth disease (HFMD) and to analyze the genetic characteristics of VP1 region in coxsackievirus B2 (CVB2) and coxsackievirus B5 (CVB5) strains circulating in Anyang area during 2011 to 2015. Methods Real-time RT-PCR and semi-nested RT-PCR were performed to identify coxsackievirus A16 (CVA16), enterovirus 71 (EV71) and other serotypes of enterovirus in order to obtain the complete etiologic composition of HFMD. The numbers of HEV-B serotypes and the percentages of specimens positive for every serotype in all enterovirus-positive specimens were calculated. As CVB2 and CVB5 were the predominant serotypes of HEV-B species, five pairs of primers targeting the VP1 regions of CVB2 and CVB5 were designed to obtain the complete nucleotide sequences of CVB2 and CVB5 VP1 regions. The phylogenetic trees were constructed based on the VP1 sequences obtained in this study and those submitted to GenBank by using MEGA7.0 and BioEdit7.2. The selection pressures on VP1 regions of CVB2 and CVB5 strains circulating in China in recent years were evaluated with the online program of DataMonkey. Results A total of 57 specimens that belonged to 14 serotypes of HEV-B species were detected in Anyang area from 2011 to 2015. The 14 serotypes of HEV-B species accounted for 56% of all serotypes of enterovirus and the specimens positive for HEV-B species accounted for 3.06% of all enterovirus-positive specimens. The HFMD cases caused by most of the HEV-B serotypes were sporadic cases. Small outbreaks of HFMD could also be caused by some serotypes of HEV-B such as CVB2 and CVB5. The complete sequences of VP1 region were obtained from 8 CVB2 strains and 9 CVB5 strains. The phylogenetic trees based on the VP1 sequences demonstrated that the CVB2 strains were classified into four genotypes (A-D). The mean evolutionary distances between different genotypes ranged from 0.191 to 0.208 and the similarities in nucleotide sequences ranged from 79.7% to 85.8%. The CVB5 strains were classified into 6 genotypes (A-F). The mean evolutionary distances and the similarities in nucleotide sequences between different genotypes of CVB5 strains ranged from 0.170 to 0.285 and 76.0% to 86.8%, respectively. Strains of different genotypes varied significantly in the residues on positons 157 and 263 in the VP1 region of CVB2 strains and on positions 75, 90 and 95 in the VP1 region of CVB5 strains. All of the CVB2 strains isolated in Anyang area belonged to D genotype and located intensively in one lineage. The CVB5 strains circulated in Anyang area belonged to F genotype and located in two lineages. The selection pressures on CVB2 strains of D genotype and CVB5 strains of F genotype circulating in China in recent years were 0.037 and 0.036, respectively. Six positively selected amino acid sites were found in the VP1 region of CVB5 strains, but no positively selected amino acid site was found in the VP1 region of CVB2 strains. Conclusion HEV-B species was an essential component of the etiologic spectrum of HFMD in Anyang area during 2011 to 2015, of which CVB5 and CVB2 were the predominant serotypes. The VP1 region of CVB5 was more complex and active than that of CVB2 over the course of evolution. Key words: Hand, foot and mouth disease; Enterovirus; Coxsackievirus B2; Coxsackievirus B5; Phylogenetic tree; Positive selection pressure

Ardisia chinensis Benth is a medicinal plant traditionally used in the area of Yao minority in Southern China. The in vitro antiviral activities of extracts and fractions from Ardisia chinensis were tested by the cytopathic effect (CPE) reduction assay in the present study. As a result, both the aqueous extract and the 95% ethanol extract of Ardisia chinensis showed in vitro antiviral activity against Coxsackie B3 (Cox B3) virus to different extents, and the aqueous extract possessed more potent activity than the ethanol extract. Bioassay-guided fractionation revealed that the antiviral activity of Ardisia chinensis was attributed mainly to its high polar fractions, and finally identified to be a polysaccharide. The Ardisia chinensis polysaccharide (ACP) fractionated from the aqueous extract exhibited a significant antiviral effect against Cox B3 with a 50% inhibitory concentration (IC(50)) of 3.9 microg/mL and a selective index (SI) over 256. Preliminary characterization indicated that ACP is a neutral polysaccharide in which d-glucose is the major component. The average molecular weights of ACP were determined to be 40037 Da (Mw), 28297 Da (Mn) and 33758 Da (Mp) by gel permeation chromatography.

Enterovirus 71 (EV71) and coxsackievirus B3 (CVB3) have resulted in severe pathogenesis caused by the host's immune response, including the cytokine cascade. Paris polyphylla Smith is a folk medicinal plant in Asia traditionally prescribed for the reduction of pain and elimination of poisoning. In this study, we investigated the anti-EV71 and CVB3 activity of P. polyphylla Smith as well as its immune modulation. The IC(50) for the P. polyphylla Smith 95% ethanol extract against EV71 and CVB3 were 12.5-23% and 99-156% of that of ribavirin, a positive control. Prevention of viral infection, viral inactivation, and anti-viral replication effects against both EV71 and CVB3 were demonstrated by the extract, the anti-viral replication effect being dominant. The extract significantly increased IL-6 production in both EV71- and CVB3-infected cells. A high correlation was possibly demonstrated between the high amounts of IL-6 induction in the EV71 and CVB3-infected cells and the anti-viral replication activity of the extract. In conclusion, good anti-EV71 and CVB3 activity was observed in the P. polyphylla Smith 95% ethanol extract. The high amounts of IL-6 induction in the virus-infected cells played a key role in the anti-viral activity of the extract.

During this study, novel biphenyl derivatives were synthesized and tested for antiviral activity. A new method based on the Suzuki coupling reaction has been established for the synthesis of these polysubstituted chain systems. In parallel with cytotoxicity, the antiviral activity of biphenyl derivatives has been determined in cytopathic effect (CPE)-inhibitory assays with the pleconaril-resistant coxsackievirus B3 (CVB3) strain Nancy, human rhinovirus 2 (HRV-2) and 14 (HRV-14) and in plaque reduction assays with the pleconaril-sensitive human isolate CVB3 97-927 in HeLa cells. Based on the results from these investigations the selectivity index (SI) was determined as the ratio of the 50% cytotoxic concentration to the 50% inhibitory concentration. The new method based on the Suzuki coupling reaction includes the condensation of 2,6-dimethyl-4-bromophenol with pentyne chloride by means of potassium carbonate and potassium iodide in N-methylpyrrolidone-2 and yields 5-bromo-1,3-dimethyl-2-(4-pentynyloxy)benzene. Its condensation with methylacetaldoxime results in 3-methylisoxazole derivatives. The following reaction with different benzeneboronic acids by means of tetrakis(triphenylphosphine)-palladium(0) finally yields the corresponding derivatives. Several of the novel synthesized derivatives demonstrated a good antiviral activity on CVB3 (SI > 2 to > 37.5) and a strong anti-HRV-2 activity (SI > 50 to > 200). In contrast, none of the compounds inhibited the HRV-14-induced CPE. These results indicate that [(biphenyloxy)propyl]isoxazole derivatives are potential inhibitors of HRV-2 and CVB3 replication, and make them promising agents for the specific treatment of these virus infections.

Antiviral activity of ginsenosides against coxsackievirus B3, enterovirus 71, and human rhinovirus 3

Synthesis and antiviral activities of a novel class of thioflavone and flavonoid analogues

Chrysin is a 5,7-dihydroxyflavone and was recently shown to potently inhibit enterovirus 71 (EV71) by suppressing viral 3C protease (3Cpro) activity. In the current study, we investigated whether chrysin also shows antiviral activity against coxsackievirus B3 (CVB3), which belongs to the same genus (Enterovirus) as EV71, and assessed its ability to prevent the resulting acute pancreatitis and myocarditis. We found that chrysin showed antiviral activity against CVB3 at 10 μM, but exhibited mild cellular cytotoxicity at 50 μM, prompting us to synthesize derivatives of chrysin to increase the antiviral activity and reduce its cytotoxicity. Among four 4-substituted benzyl derivatives derived from C(5) benzyl-protected derivatives 7, 9–11 had significant antiviral activity and showed the most potent activity against CVB3 with low cytotoxicity in Vero cells. Intraperitoneal injection of CVB3 in BALB/c mice with 1×106 TCID50 (50% tissue culture infective dose) of CVB3 induced acute pancreatitis with ablation of acinar cells and increased serum CXCL1 levels, whereas the daily administration of 9 for 5 days significantly alleviated the pancreatic inflammation and reduced the elevation in serum CXCL1 levels. Collectively, we assessed the anti-CVB3 activities of chrysin and its derivatives, and found that among 4-substituted benzyl derivatives, 9 exhibited the highest activity against CVB3 in vivo, and protected mice from CVB3-induced pancreatic damage, simultaneously lowering serum CXCL1 levels.

Ursolic acid (UA), a pentacyclic triterpenoid acid found in many medicinal plants and aromas, is known for its antibacterial effects against multi-drug-resistant (MDR) Gram-positive bacteria, which seriously threaten human health. Unfortunately, UA water-insolubility, low bioavailability, and systemic toxicity limit the possibilities of its application in vivo. Consequently, the beneficial activities of UA observed in vitro lose their potential clinical relevance unless water-soluble, not cytotoxic UA formulations are developed. With a nano-technologic approach, we have recently prepared water-soluble UA-loaded dendrimer nanoparticles (UA-G4K NPs) non-cytotoxic on HeLa cells, with promising physicochemical properties for their clinical applications. In this work, with the aim of developing a new antibacterial agent based on UA, UA-G4K has been tested on different strains of the Enterococcus genus, including marine isolates, toward which UA-G4K has shown minimum inhibitory concentrations (MICs) very low (0.5–4.3 µM), regardless of their resistance to antibiotics. Time-kill experiments, in addition to confirming the previously reported bactericidal activity of UA against E. faecium, also established it for UA-G4K. Furthermore, cytotoxicity experiments on human keratinocytes revealed that nanomanipulation of UA significantly reduced the cytotoxicity of UA, providing UA-G4K NPs with very high LD50 (96.4 µM) and selectivity indices, which were in the range 22.4–192.8, depending on the enterococcal strain tested. Due to its physicochemical and biological properties, UA-G4K could be seriously evaluated as a novel oral-administrable therapeutic option for tackling difficult-to-treat enterococcal infections.

Coxsackievirus infections are associated with cases of aseptic meningitis, encephalitis, myocarditis, and some chronic disease. A series of benzo[d][1,2,3]triazol-1(2)-yl derivatives (here named benzotriazol-1(2)-yl) (4a-i, 5a-h, 6a-e, g, i, j and 7a-f, h-j) were designed, synthesized and in vitro evaluated for cytotoxicity and antiviral activity against two important human enteroviruses (HEVs) members of the Picornaviridae family [Coxsackievirus B 5 (CVB-5) and Poliovirus 1 (Sb-1)]. Compounds 4c (CC50 >100 μM; EC50 = 9 μM), 5g (CC50 >100 μM; EC50 = 8 μM), and 6a (CC50 >100 μM; EC50 = 10 μM) were found active against CVB-5. With the aim of evaluating the selectivity of action of this class of compounds, a wide spectrum of RNA (positive- and negativesense), double-stranded (dsRNA) or DNA viruses were also assayed. For none of them, significant antiviral activity was determined. These results point towards a selective activity against CVB-5, an important human pathogen that causes both acute and chronic diseases in infants, young children, and immunocompromised patients.

Cepharanthine (CEP) is a natural bisbenzylisoquinoline alkaloid with broad-spectrum antiviral activity. However, the extremely poor water solubility severely limits the pharmaceutical development and in vivo application of CEP. This study aimed to identify safe and effective solubilizing systems to enhance the solubility of CEP and systematically assess how these changes affect the inhibitory activity of CEP against coronaviruses. The solubility of CEP in various natural acidic media, including Rosa roxburghii juice, vinegar, and apple cider vinegar, as well as in solubilizing systems such as vitamin C and glycyrrhizic acid, was systematically investigated. The GX_P2V coronavirus infection model was used to measure the antiviral activity (EC50), cytotoxicity, and selectivity index (SI) of CEP under different solubilization conditions. Additionally, hydrochloride, pyruvate, and mesylate salts of CEP were prepared through acid-base neutralization and tested for their in vitro antiviral activity. The results showed that the solubility of CEP was significantly improved under acidic conditions with pH 2‒4. A mixture of vitamin C and CEP at a mass ratio of 4:1 formed a clear solution and demonstrated enhanced antiviral activity (EC50=0.85 μmol/L, SI=17.01) compared with CEP alone. Although glycyrrhizic acid increased the solubility of CEP in 30% ethanol, it exhibited poor water stability and did not significantly enhance the antiviral activity. Among all tested systems, CEP hydrochloride showed the best antiviral selectivity (EC50=1.05 μmol/L, SI>47). In conclusion, both the vitamin C-based formulation and CEP hydrochloride significantly improved the solubility and antiviral activity of CEP, highlighting its potential for further pharmaceutical development and clinical application.

Preparation of new series of 1-[4-(5-Phenyl-4,5 dihydro-1H-pyrazole-3-yl)-phenyl]-pyrrole-2,5-dione and 1-[4- (5-Phenyl-4,5 dihydroisooxazole-3-yl)-phenyl]-pyrrole-2,5-dione derivatives, derived from α,β-unsaturated ketones with objective of obtaining lead compounds for future development as antiviral agents. The inhibition of virus-induced cytopathicity or plaque formation in HEL [herpes simplex virus type 1 (HSV-1) (KOS), HSV-2 (G), vaccinia virus, vesicular stomatitis virus, cytomegalovirus (HCMV), and varicella-zoster virus (VZV)], Vero (parainfluenza-3, reovirus-1, Sindbis virus and Coxsackie B4), HeLa (vesicular stomatitis virus, Coxsackie virus B4, and respiratory syncytial virus) or MDCK [influenza A (H1N1; H3N2) and influenza B] cell cultures by synthesized derivatives. All the compounds were characterized by physical, spectroscopic and elemental analysis. Among the fourteen synthesized targets only compounds 3g and 4e showed activity against RSV (EC50 = 37 µM and 46 µM with affecting cell morphology at concentrations of, respectively, 254 µM and 286 µM) Compound 3g also showed weak activity against feline corona virus (EC50 value of 29 µM and a MCC of 107 µM) for CRFK cells. Feline corona virus was also weakly inhibited by compound 3f (EC50=39 µM) that altered cell morphology at a concentration of 231 µM. Except for feline corona virus, none of the other viruses was inhibited by compound 3f. Low selectivity indices (SIs), defined as ratio MCC/EC50, were observed SI=4 and SI=6 for compounds 3g and 3f against feline corona virus respectively, while SI=6 for compound 4e against RSV, and SI=7 for compound 3g against RSV. Biological evaluation was performed using acyclovir, brivudin, gancilcovir, cidofovir against herpes and poxviruses while ribavirin against RNA viruses, reference standard drugs. Keywords: Antiviral activity, 4, 5 dihydro-1H-pyrazoles, 4, 5 dihydroisoxazoles.

The aim of the present work was to carry out a bioguided isolation of antiviral chemical constituents from an ethanol extract of leaves from Arrabidaea pulchra (Cham.) Sandwith (EEAPL) that had shown in vitro activity in a previous screening using DNA and RNA viruses. The activity of EEPAL was evaluated against the DNA viruses Human herpesvirus 1 (HSV-1) and Vaccinia virus Western Reserve (VACV-WR) as well as against the RNA viruses Murine encephalomyocarditis virus (EMCV), and Dengue virus 2 (DENV-2) by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. Cytotoxicity was determined in LLCMK2 and Vero cells and the Selectivity Indexes (SI) were calculated. The most potent effect was observed against DENV-2 (EC50 46.8 ± 1.6 µg mL−1; SI 2.7). For HSV-1 and VACV-WR EC50 values > 200 µg mL−1 were determined, while no inhibition of the cytopathic effect was observed with EMCV. Bioguided fractionation of EEAPL by partition between immiscible solvents followed by chromatography over a Sephadex LH20 column afforded two arylpropanoid glycosides, verbascoside (AP 1) and caffeoylcalleryanin (AP 2), along with a terpenoid, ursolic acid (AP 3). AP 1 and AP 3 exhibited similar anti-DENV-2 profiles, with SI values of 3.8 and 3.1, respectively, while AP 2 was the most effective anti-DENV-2 constituent, with a SI of 20.0. Our results show that A. pulchra leaves ethanol extract (EEAPL) affords compounds with antiviral activity, mainly against DENV-2.

Leishmaniasis is a neglected tropical disease caused by the flagellated protozoa of the genus Leishmania that affects millions of people around the world. Drugs employed in the treatment of leishmaniasis have limited efficacy and induce local and systemic side effects to the patients. Natural products are an interesting alternative to treat leishmaniasis, because some purified molecules are selective toward parasites and not to the host cells. Thus, the aim of the present study was to compare the in vitro antileishmanial activity of the triterpenes betulin (Be), lupeol (Lu), and ursolic acid (UA); analyze the physiology and morphology of affected organelles; analyze the toxicity of selected triterpenes in golden hamsters; and study the therapeutic activity of triterpenes in hamsters infected with L. (L.) infantum as well as the cellular immunity induced by studied molecules. The triterpenes Lu and UA were active on promastigote (IC50 = 4.0 ± 0.3 and 8.0 ± 0.2 μM, respectively) and amastigote forms (IC50 = 17.5 ± 0.4 and 3.0 ± 0.2 μM, respectively) of L. (L.) infantum, and their selectivity indexes (SI) toward amastigote forms were higher (≥13.4 and 14, respectively) than SI of miltefosine (2.7). L. (L.) infantum promastigotes treated with Lu and UA showed cytoplasmic degradation, and in some of these areas, cell debris were identified, resembling autophagic vacuoles, and parasite mitochondria were swelled, fragmented, and displayed membrane potential altered over time. Parasite cell membrane was not affected by studied triterpenes. Studies of toxicity in golden hamster showed that Lu did not alter blood biochemical parameters associated with liver and kidney functions; however, a slight increase of aspartate aminotransferase level in animals treated with 2.5 mg/kg of UA was detected. Lu and UA triterpenes eliminated amastigote forms in the spleen (87.5 and 95.9% of reduction, respectively) and liver of infected hamster (95.9 and 99.7% of reduction, respectively); and UA showed similar activity at eliminating amastigote forms in the spleen and liver than amphotericin B (99.2 and 99.8% of reduction). The therapeutic activity of both triterpenes was associated with the elevation of IFN-γ and/or iNOS expression in infected treated animals. This is the first comparative work showing the in vitro activity, toxicity, and therapeutic activity of Lu and UA in the chronic model of visceral leishmaniasis caused by L. (L.) infantum; additionally, both triterpenes activated cellular immune response in the hamster model of visceral leishmaniasis.